Glucan, water dikinase activity stimulates breakdown of starch granules by plastidial β-amylases
Glucan, water dikinase activity stimulates breakdown of starch granules by plastidial β-amylases
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DOI：
10.1104/pp.107.104224
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发表时间：
2007-09-01
期刊：
影响因子：
7.4
通讯作者：
Ritte, Gerhard
中科院分区：
文献类型：
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作者：
Edner, Christoph;Li, Jing;Ritte, Gerhard
葡聚糖磷酸化酶是拟南芥（Arabidopsis thaliana）和马铃薯（Solanum tuberosum）叶片中淀粉正常动员所必需的，但这种依赖性的机制尚不清楚。使用两种不同的活性测定，我们的目的是确定淀粉降解酶从拟南芥，其活性的葡聚糖磷酸化的影响。从叶提取物中纯化的蛋白质组分颗粒淀粉的分解增加了约2倍，如果颗粒同时被重组马铃薯葡聚糖，水双激酶（GWD）磷酸化。使用基质辅助激光解吸电离质谱法在该馏分中鉴定了几种推定的淀粉相关酶，其中包括β-淀粉酶1（BAM 1; At 3g 23920）和异淀粉酶3（ISA 3; At 4g 09020）。使用纯化的重组酶的实验表明，BAM 1活性与颗粒类似地增加的条件下，同时淀粉磷酸化。纯化的重组马铃薯ISA 3（StISA 3）没有攻击颗粒淀粉显着与或不与葡聚糖磷酸化。然而，由BAM 1和StISA 3的混合物的淀粉分解是由BAM 1单独的2倍，并在GWD和ATP的存在下进一步增强。与BAM 1类似，如果颗粒同时被GWD磷酸化，则通过纯化的重组BAM 3（At 4g 17090）（另一种质体定位的β-淀粉酶同种型）从颗粒淀粉中释放的麦芽糖增加2至3倍。BAM活性反过来强烈刺激GWD催化的磷酸化。GWD和BAM活性之间的相互依赖性为GWD缺陷突变体的严重淀粉过量表型提供了解释。
Glucan phosphorylating enzymes are required for normal mobilization of starch in leaves of Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum), but mechanisms underlying this dependency are unknown. Using two different activity assays, we aimed to identify starch degrading enzymes from Arabidopsis, whose activity is affected by glucan phosphorylation. Breakdown of granular starch by a protein fraction purified from leaf extracts increased approximately 2-fold if the granules were simultaneously phosphorylated by recombinant potato glucan, water dikinase (GWD). Using matrix-assisted laser-desorption ionization mass spectrometry several putative starch-related enzymes were identified in this fraction, among them beta-AMYLASE1 (BAM1; At3g23920) and ISOAMYLASE3 (ISA3; At4g09020). Experiments using purified recombinant enzymes showed that BAM1 activity with granules similarly increased under conditions of simultaneous starch phosphorylation. Purified recombinant potato ISA3 (StISA3) did not attack the granular starch significantly with or without glucan phosphorylation. However, starch breakdown by a mixture of BAM1 and StISA3 was 2 times higher than that by BAM1 alone and was further enhanced in the presence of GWD and ATP. Similar to BAM1, maltose release from granular starch by purified recombinant BAM3 (At4g17090), another plastid-localized beta-amylase isoform, increased 2-to 3-fold if the granules were simultaneously phosphorylated by GWD. BAM activity in turn strongly stimulated the GWD-catalyzed phosphorylation. The interdependence between the activities of GWD and BAMs offers an explanation for the severe starch excess phenotype of GWD-deficient mutants.